CN104478783A - Synthesis method of compound for protein fluorescent labeling - Google Patents
Synthesis method of compound for protein fluorescent labeling Download PDFInfo
- Publication number
- CN104478783A CN104478783A CN201410592878.9A CN201410592878A CN104478783A CN 104478783 A CN104478783 A CN 104478783A CN 201410592878 A CN201410592878 A CN 201410592878A CN 104478783 A CN104478783 A CN 104478783A
- Authority
- CN
- China
- Prior art keywords
- compound
- equivalent
- probe
- synthetic method
- fluorescent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a synthesis method of a compound for protein fluorescent labeling and relates to the technical field of biomacromolecule fluorescent labeling. The synthesis method comprises the following steps of 1, reaction 1: heating 1.2-1.5 equivalent of a compound I and 1 equivalent of a compound II in 1.5-3.5 equivalent of acetic anhydride with stirring for 0.5-1h to obtain an intermediate III, and 2, reaction 2: heating 1 equivalent of the intermediate III and 1.1-1.5 equivalent of sodium iodide in an organic solvent 2 to a temperature of 40-70 DEG C, and carrying out reflux for 2-24h to obtain a desired product IV which is a water-soluble fluorescent compound. A cyanine compound is introduced with multiple hydrophilic sulfonic acid groups so that water solubility is improved. A distance between a connection locus and a fluorescent group is further reduced so that probe rigidity is improved and thus the problem that the existing protein fluorescent probe has nonuniform hydrophobic ends and hydrophilic ends and has long length of a probe connection group.
Description
Technical field:
The present invention relates to the fluorescent labelling techniques field of biomacromolecule, be specifically related to a kind of synthetic method of the compound for protein fluorescence mark.
Background technology:
The research method of biomacromolecule dynamics comprises nucleus magnetic resonance, X-ray crystalline diffraction, Small angle X-ray scattering, FRET (fluorescence resonance energy transfer) etc.Wherein, fluorescent method because its detection sensitivity is high, the advantage such as experiment is convenient, be one of research means the most widely.
Fluorescence Resonance Energy transfer is a kind of energy transfer phenomenon apart from producing between two very near fluorescence molecules.When the emmission spectrum of donor fluorescent molecule is overlapping with the absorption spectrum of acceptor fluorescence molecule, and when the distance of two molecules is within 10nm scope, a kind of inactive energy trasfer will be there is, i.e. FRET phenomenon, many (quenching of fluorescence) that will be low when making its Individual existence of the fluorescence intensity ratio of donor, and the fluorescence of acceptor emission strengthens (sensitized fluorescence) greatly.
The products such as Cy3-NHS-ester, Cy3Maleimide of GE company are comprised at the commercialization product of the fluorescent probe of 500-600nm in excitation wavelength, but these product water dissolubilities are poor, be unfavorable for marking the macromole in the aqueous solution, and the carbochain connected is longer, its larger flexibility is unfavorable for accurately describing the Structure and dynamics feature of biomacromolecule.And the people such as Alexei Toutchkine to have developed a series of compound such as Cy3.2-IA, neo-Cy3 as shown below, though have water-soluble preferably, but due to the hydrophilic of compound and hydrophobic grouping skewness, there is obvious water-wet side and hydrophobic side, make probe skewness in the solution, thus affect the accuracy of experimental result.
Summary of the invention:
The object of this invention is to provide a kind of synthetic method of the compound for protein fluorescence mark, the compound of flower cyanines element class is introduced multiple hydrophilic sulfonic acid group by it, improves water-soluble; Further reduce the distance of connection site and fluorophor, improve the rigidity of probe; Thus solve the longer problem of current protein fluorescence probe water-wet side and hydrophobic side skewness, probe linker.
In order to solve the problem existing for background technology, the present invention is by the following technical solutions: its synthetic method is divided into two steps, the first step reaction one is the Compound II per heated and stirred 0.5-1 hour in the acetic anhydride of 1.5-3.5 equivalent by the Compound I of 1.2-1.5 equivalent and 1 equivalent, obtains intermediate product III; Second step reaction two is that the intermediate product III of 1 equivalent and the sodium iodide of 1.1-1.5 equivalent are heated to the 40-70 DEG C of 2-24 hour that refluxes in organic solvent 2, obtains target product IV, i.e. water soluble fluorescence compound.
In described Compound I structure, corresponding positively charged ion is potassium ion or sodium ion.
In described Compound II per structure, corresponding positively charged ion is potassium ion or sodium ion.
Described organic solvent 1 is the mixed solvent of methyl alcohol and chloroform or the mixed solvent of methyl alcohol and methylene dichloride.
Advantage of the present invention is: the compound of flower cyanines element class is introduced multiple hydrophilic sulfonic acid group by probe of the present invention, improves water-soluble; Further reduce the distance of connection site and fluorophor, improve the rigidity of probe; Thus solve the longer problem of current protein fluorescence probe poorly water-soluble, hydrophilic radical and hydrophobic grouping skewness, linking group.The probe that this fluorescent chemicals marks as a kind of protein sulfhydryl, effectively can reduce background fluorescence interference, improve signal to noise ratio.Meanwhile, the flexibility of this fluorescent probe is less, is conducive to the accurate description of the Structure and dynamics feature to biomacromolecule.Simple synthetic method, the toxicity of fluorescent chemicals of the present invention are less.
Accompanying drawing illustrates:
Fig. 1 is background technology accompanying drawing of the present invention;
Fig. 2 is the structural representation of middle probe of the present invention;
Fig. 3 is the structural representation of the present invention two kinds reaction;
Fig. 4 is excitation-emission collection of illustrative plates of the present invention.
Embodiment:
Referring to Fig. 1-Fig. 4, this embodiment is by the following technical solutions: its synthetic method is divided into two steps, the first step reaction one is the Compound II per heated and stirred 0.5-1 hour in the acetic anhydride of 1.5-3.5 equivalent by the Compound I of 1.2-1.5 equivalent and 1 equivalent, obtains intermediate product III; Second step reaction two is that the intermediate product III of 1 equivalent and the sodium iodide of 1.1-1.5 equivalent are heated to the 40-70 DEG C of 2-24 hour that refluxes in organic solvent 2, obtains target product IV, i.e. water soluble fluorescence compound.
In described Compound I structure, corresponding positively charged ion is potassium ion or sodium ion.
In described Compound II per structure, corresponding positively charged ion is potassium ion or sodium ion.
Described organic solvent 1 is the mixed solvent of methyl alcohol and chloroform or the mixed solvent of methyl alcohol and methylene dichloride.
The advantage of this embodiment is: the compound of flower cyanines element class is introduced multiple hydrophilic sulfonic acid group by probe of the present invention, improves water-soluble; Further reduce the distance of connection site and fluorophor, improve the rigidity of probe; Thus solve the longer problem of current protein fluorescence probe poorly water-soluble, hydrophilic radical and hydrophobic grouping skewness, linking group.The probe that this fluorescent chemicals marks as a kind of protein sulfhydryl, effectively can reduce background fluorescence interference, improve signal to noise ratio.Meanwhile, the flexibility of this fluorescent probe is less, is conducive to the accurate description of the Structure and dynamics feature to biomacromolecule.Simple synthetic method, the toxicity of fluorescent chemicals of the present invention are less.
The embodiment of this embodiment:
One, for a compound for protein fluorescence mark, the method follows these steps to carry out:
Get the sodium salt of the sylvite of 1 equivalents of compound I and the Compound II per of 1.2 equivalents, add the sym-dichloroacetic anhydride of 1.5 equivalents, stirring at room temperature 0.5 hour.Revolve and steam except desolventizing, column chromatography, with methyl alcohol: acetone=1: 7 wash-outs, obtains corresponding compound III.By the compound III of 1 obtained equivalent and the sodium iodide of 1.1 equivalents at methyl alcohol: chloroform=1: in the solvent of 1,60 DEG C are reacted 24 hours, filter, revolve and steam except desolventizing, obtain corresponding final product IV.Confirm to coincide with target product through nuclear-magnetism spectrum.
Two, for a synthetic method for the water-soluble near infrared fluorescent probe of protein labeling, the method follows these steps to carry out:
Get the sodium salt of the sodium salt of the Compound I of 1 equivalent and the Compound II per of 1.2 equivalents, add the sym-dichloroacetic anhydride of 2 equivalents, heated and stirred 0.7 hour.Revolve and steam except desolventizing, column chromatography, with methyl alcohol: acetone=1: 5 wash-outs, obtains corresponding compound III.By the compound III of 1 obtained equivalent and the sodium iodide of 1.5 equivalents at methyl alcohol: chloroform=1: in the solvent of 2,70 DEG C are reacted 20 hours, filter, revolve and steam except desolventizing, obtain corresponding final product IV.Coincide with target product through nuclear-magnetism display.
Three, for a synthetic method for the water-soluble near infrared fluorescent probe of protein labeling, the method follows these steps to carry out:
Get the sylvite of the Compound II per of sylvite 0.4g and the 0.6g of Compound I, the molten acetic acid adding 0.2g, heated and stirred 1 hour.Revolve and steam except desolventizing, column chromatography, with methyl alcohol: acetone=1: 5 wash-outs, obtains corresponding compound III.By the compound III of obtained 0.4g and the sodium iodide of 0.1g at methyl alcohol: chloroform=1: in the solvent of 10,65 DEG C are reacted 24 hours, filter, revolve and steam except desolventizing, obtain corresponding final product IV 0.40g.Show through nuclear-magnetism and mass spectrum and coincide with target product.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (4)
1. the synthetic method for the compound of protein fluorescence mark, it is characterized in that it comprises synthetic method and is divided into two steps, the first step reaction one is the Compound II per heated and stirred 0.5-1 hour in the acetic anhydride of 1.5-3.5 equivalent by the Compound I of 1.2-1.5 equivalent and 1 equivalent, obtains intermediate product III; Second step reaction two is that the intermediate product III of 1 equivalent and the sodium iodide of 1.1-1.5 equivalent are heated to the 40-70 DEG C of 2-24 hour that refluxes in organic solvent 2, obtains target product IV, i.e. water soluble fluorescence compound.
2. the synthetic method of a kind of compound for protein fluorescence mark according to claim 1, it is characterized in that in described Compound I structure, corresponding positively charged ion is potassium ion or sodium ion.
3. the synthetic method of a kind of compound for protein fluorescence mark according to claim 1, it is characterized in that in described Compound II per structure, corresponding positively charged ion is potassium ion or sodium ion.
4. the synthetic method of a kind of compound for protein fluorescence mark according to claim 1, is characterized in that described organic solvent 1 is the mixed solvent of methyl alcohol and chloroform or the mixed solvent of methyl alcohol and methylene dichloride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410592878.9A CN104478783A (en) | 2014-10-30 | 2014-10-30 | Synthesis method of compound for protein fluorescent labeling |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410592878.9A CN104478783A (en) | 2014-10-30 | 2014-10-30 | Synthesis method of compound for protein fluorescent labeling |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104478783A true CN104478783A (en) | 2015-04-01 |
Family
ID=52753418
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410592878.9A Pending CN104478783A (en) | 2014-10-30 | 2014-10-30 | Synthesis method of compound for protein fluorescent labeling |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104478783A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108840815A (en) * | 2018-05-29 | 2018-11-20 | 苏州百源基因技术有限公司 | A kind of amino acid of fluorescent marker and its preparation method and application |
| CN110204562A (en) * | 2019-07-17 | 2019-09-06 | 大连理工大学 | One kind has fluorescent chemicals and the application of protein labeling function |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008027075A2 (en) * | 2006-02-16 | 2008-03-06 | The Scripps Research Institute | Sulfhydryl-reactive, water soluble dyes |
| CN103952142A (en) * | 2014-03-04 | 2014-07-30 | 中国科学院武汉物理与数学研究所 | Water-soluble near-infrared fluorescent probe used for protein labeling and synthetic method thereof |
| CN103952143A (en) * | 2014-03-04 | 2014-07-30 | 中国科学院武汉物理与数学研究所 | Trimethyl cyanine fluorescence probe used for protein labeling and synthetic method thereof |
-
2014
- 2014-10-30 CN CN201410592878.9A patent/CN104478783A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008027075A2 (en) * | 2006-02-16 | 2008-03-06 | The Scripps Research Institute | Sulfhydryl-reactive, water soluble dyes |
| CN103952142A (en) * | 2014-03-04 | 2014-07-30 | 中国科学院武汉物理与数学研究所 | Water-soluble near-infrared fluorescent probe used for protein labeling and synthetic method thereof |
| CN103952143A (en) * | 2014-03-04 | 2014-07-30 | 中国科学院武汉物理与数学研究所 | Trimethyl cyanine fluorescence probe used for protein labeling and synthetic method thereof |
Non-Patent Citations (2)
| Title |
|---|
| ALEXEI TOUTCHKINE等: "Facile Synthesis of Thiol-Reactive Cy3 and Cy5 Derivatives with Enhanced Water Solubility", 《BIOCONJUGATE CHEMISTRY》 * |
| ALEXEI TOUTCHKINE等: "Simple One-Pot Preparation of Water-Soluble, Cysteine-Reactive Cyanine and Merocyanine Dyes for Biological Imaging", 《BIOCONJUGATE CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108840815A (en) * | 2018-05-29 | 2018-11-20 | 苏州百源基因技术有限公司 | A kind of amino acid of fluorescent marker and its preparation method and application |
| CN110204562A (en) * | 2019-07-17 | 2019-09-06 | 大连理工大学 | One kind has fluorescent chemicals and the application of protein labeling function |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105542756B (en) | A kind of fluorescence probe for detecting formaldehyde and preparation method and application | |
| Krasnoperov et al. | Luminescent probes for ultrasensitive detection of nucleic acids | |
| Dong et al. | A novel ferrocenyl-based multichannel probe for colorimetric detection of Cu (II) and reversible fluorescent “turn-on” recognition of Hg (II) in aqueous environment and living cells | |
| CN110234735B (en) | Bimodal biological imaging probe | |
| US9139868B2 (en) | Silicon and germanium dyes for use in genetic identity | |
| CN107226783B (en) | A kind of lysosome targeting fluorescent probe and preparation method thereof | |
| Xing et al. | A novel aggregation induced emission (AIE) fluorescence probe by combining tetraphenylethylene and 2′, 3′-O-isopropylideneadenosine for localizing Golgi apparatus | |
| Bura et al. | Fluorescent boron dipyrromethene (Bodipy) dyes having two and four vinyl residues | |
| WO2020221217A1 (en) | Fluorescent dye, preparation method therefor, and use thereof | |
| Wang et al. | First Synthesis of an Oridonin‐Conjugated Iridium (III) Complex for the Intracellular Tracking of NF‐κB in Living Cells | |
| CN102887914B (en) | Coumarins compound containing aryl boric acid and application thereof in detection of sugar | |
| Yang et al. | BODIPY derivatives bearing borneol moieties: Enhancing cell membrane permeability for living cell imaging | |
| Qu et al. | Molecular rotor based on dipyridylphenylamine: Near-infrared enhancement emission from restriction of molecular rotation and applications in viscometer and bioprobe | |
| CN102443018B (en) | Fluorescence-labeled O6-benzyl guanine and preparation and application thereof | |
| CN104151867B (en) | Temperature response type cyclodextrin probe double with pH and preparation method thereof | |
| CN102154003B (en) | Novel carbazole-bridge-based fluorescent cyanine dye probe and preparation method thereof | |
| Li et al. | Recent advances in N-heterocyclic small molecules for synthesis and application in direct fluorescence cell imaging | |
| CN104478783A (en) | Synthesis method of compound for protein fluorescent labeling | |
| CN102516987A (en) | Fluorescent probe for detecting nitrogen monoxide and preparation method thereof | |
| Wei et al. | Adenine-based small molecule fluorescent probe for imaging mitochondrial nucleic acid | |
| Kim et al. | Imidazolyl–benzocoumarins as ratiometric fluorescence probes for biologically extreme acidity | |
| Wu et al. | Fluorescent hydrogen sulfide probes based on azonia-cyanine dyes and their imaging applications in organelles | |
| Kwon et al. | Indolizino [3, 2-c] quinolines as environment-sensitive fluorescent light-up probes for targeted live cell imaging | |
| Xu et al. | A mitochondria-targeted fluorescent probe based on biocompatible RBH-U for the enhanced response of Fe3+ in living cells and quenching of Cu2+ in vitro | |
| Yu et al. | Carbazole-based fluorescent probes for G-quadruplex DNA targeting with superior selectivity and low cytotoxicity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150401 |